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CHAPTER Ι
INTRODUCTION
Fluorescent correlation spectroscopy is commonly used technique to study the
dynamics of molecules due to its high sensitivity, it also can be employed in biomedical,
chemical, and biological fields(Weiss, 1999).Since the establishment of very sensitive
detector such as avalanche photodiodes, ready to use ultrafast laser sources which are in the
NIR,MAIL from Au, NP can now be often exploited so that they can track these
nanoparticles in vitro and in vivo biological experiments (Webb & Bardhan, 2014).
Gold nanostructures attract particular interest because of their favorable emission
properties in terms of good photo stability under continuous irradiation, there are no blinking
effects, and good biocompatibility (exact with respect to the potential leaching of toxic metal
ions from semiconductor quantum dots). The likelihood of tuning the absorption band by
modifying the particles shape is also another advantage for their application in different
fields. A detailed investigation on the luminescence behavior, under one- and two-photon
excitation at increasing laser power, of citrate capped gold nanoparticles (Au NP) with
increasing diameter (up to 50 nm) has been reported by Loumaigne and Colleagues.
According to the fluorescence correlation spectroscopy (FCS ) analysis over the past
years researchers found that there is a linear relationship between the diameters of gold Nano
spheres and the diffusion time. They also found that the size, shape, and surrounding surface
environment are crucial in the optical properties of nanoparticles
In addition to that the nanoparticles showed high photo-saturation feature and
increased brightness per particle when excited with strong laser light. In accordance with all
the above mentioned reasons, the nanotechnology becomes widely used in medical
applications like drug delivery and specifically the photodynamic therapy and diagnosis (De
Jong, 2008).
FCS experiments are run under increasing the laser power, using different samples of
colloidal gold solutions, using different wavelengths, and using different solutions to be
dispersed in. A review for the fluorescence correlation spectroscopy, nanoparticles,
photodynamictherapy, and discussion for the results will be presented in the following
sections of this write-up.
There has been a high growth of research and applications in the area of Nano science
as well as nanotechnology in the past years. Recently there is an increasing optimism that
nanotechnology while applied to medicine will bring important developments in the
diagnosis as well as the treatment of the disease. Applications in medicine that are anticipated
are such as drug delivery, vitro and vivo diagnostic, production of improved biocompatible
materials as well as nutraceuticals. Engineered nanoparticles are an important tool since they
help one to realize a number of these applications.
It has also been identified that not all particles that are used for medical purposes
comply with the acceptable definition that has been recently proposed by the Royal Society
and Royal Academy of Engineering of a size 100nm.This has not proven to have any impact
on the functionality of medical applications. Moreover for drug delivery not only engineered
particles may be used as carrier. The drug may be formulated on itself at a nano scale and
then function on itself as a carrier. Source materials may be of biological origin or have
biological origin such as lipids or lactic acids.
Correlation
Correlation is referred to as a statistical measurement, which is utilized to describe the
relation between two fluctuating signals (Cross-Correlation) or the signal with itself
(Autocorrelation). On the other hand, correlation can be defined in different ways according
to the field of study. In essence, the correlation being a statistical measure portrays the
relationship and degree to which two variables or more fluctuate together. In signal
processing, the correlation used to analyze functions or series of values like the time domain
signals. Subsequently, correlation is mainly defined in two terminologies, which are a
positive correlation and a negative correlation (Rigler & Elson, 2012).
Hence, a positive correlation shows the level at which variables decrease or increase
in parallel. On the other hand, a negative correlation shows the level at which one of the
variables increases while the other variable decreases. Cross-Correlation in signal processing
refers to a similarity measure of two series mainly as a lag function of one relative series to
the other. (Berezin, 2014).
Microscopy
Microscopy refers to a noble scientific practice, which comprises of magnifying
objects that the unaided eye cannot see. Thus, the main objective of this scientific discipline
is to be able to magnify the object so that it is visible for studying. This allows researchers to
conduct their study and learn essential things about the invisible objects, as well as how they
work. In addition, microscopy utilizes microscopes to view these objects and samples. There
are mainly three major branches of microscopy, which include scanning probe, electron and
optical microscopy (Berberan-Santos, 2008).
On the same note, electron and optical microscopy involve the refraction, reflection or
diffraction of electromagnetic electron/radiation beams that interact with the specimen. It also
interacts with the scattered radiation collection or any other signal so that it can create an
image. Conversely, this process can be performed through the sample wide-field irradiation
or through scanning the sample’s fine beam. Scanning probe microscopy comprises of the
scanning probe interaction with the sample’s surface (Berezin, 2014).
Confocal Microscopy
Confocal Microscopy refers to an optical imaging method, which is used to increase
the optical resolution and the difference of a micrograph through an additional spatial pinhole
that is placed on the lens’ confocal plane. Confocal Microscopy has gradually gained
popularity particularly in the industrial and scientific communities. Its distinctive applications
are in materials science, life sciences and semiconductor (Rigler & Elson, 2012).
In this sense, this technique provides numerous advantages as compared to the
conventional optical microscopy. These advantages include out-of-focus glare elimination,
narrow depth of field and its ability to gather serial optical parts from thick specimens (Rege
& Medintz, 2009).
Fluorophores
A fluorophore is also known as a fluorochrome, and it is similar to a chromophore. A
fluorophore is mainly a fluorescent chemical substance, which can re-emit light when light
excitation takes place. Hence, a fluorophore refers to a section of a molecule, which leads to the
creation of a fluorescent emission specifically in the observable light spectrum. These
fluorophores absorb different light wavelengths, and this creates the visible light. These
fluorophores can be introduced through artificial methods, or they can exist naturally. It is
paramount to note that many rocks and fish maintain some natural levels of these fluorophores
(Berberan-Santos, 2008).
Nevertheless, the fluorophores are utilized widely in the scientific fraternity for research
purposes since they assist in analyzing certain material properties. Hence, researchers can
identify changes and reactions in the biochemistry fields, as well as protein study. Besides, the
immunofluorescence discipline utilizes this technique to assist in labeling antibodies and
antigens at the level of subcellular (Rege & Medintz, 2009).
LASERs
The acronym LASER stands for “Light Amplification by Stimulated Emission of
Radiation.” Theodore Maiman invented it in 1964. The LASER device can produce
monochromatic, directive, and coherence light, which could serve many useful inventions.
LASER device composed of external power supply, cavity, and active medium. LASERs can be
classified into (solid, gas, liquid, excimer, chemical, and semiconductor) LASERs depending on
the active medium or the lasing material. A LASER is different from other light sources because
it emits its light coherently whereby spatial coherence enables it to be focused to one tight spot.
Ideally, this enables various applications such as lithography and laser cutting (Rege & Medintz,
2009).
In principle, this technique has many applications and can be used in laser printers, laser
surgery, barcode scanners, optical disk drives, free-space and fiber-optic optical communication.
Others include being used in skin treatments, laser lighting displays, welding and cutting
materials (Berberan-Santos, 2008).
Stokes Shift
When absorbing light , the atom or the molecule undergoes a transition into an excited
electronic state accompanies with losing small amount of absorbing energy before releasing the
rest of its energy as luminescence , thermal energy in most cases,. The difference between the
band maxima of the absorption and luminescence spectra within the same electronic state is
known as Stokes shift and it could be represented in frequency or wavelength units.
Fig.(?): Stokes
shift (Fanucci, 2014).
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy (FCS) is a correlation of temporal fluctuations of
the fluorescence intensity.
Fig.(?) : Conceptual diagram of a fluctuating fluorescence signal (A) as a function of time. The fluctuation
of the signal is used to calculate the autocorrelation, G(τ), where τ is the lag time from the original signal. The
amplitude of the autocorrelation function (typically G(0)) is inversely proportional to the average number of
molecules in the probe volume (), (NCBI.gov, Figure 1).
F
CS
is
one
of
the
ma
ny different modes of high resolution spatial and temporal analysis of extremely low
concentrated biomolecules.FCS relays information on the photo-physics that cause
characteristics of absolute concentration of detected particles, diffusion behaviour of detected
particles and fluorescence intensity fluctuation. In general, all the physical parameters that give
rise to fluctuations in the fluorescence signal are accessible by FCS (Rigler& Elson, 2001).
FCS measures the fluctuations of fluorescence intensity in a sub-femtolitre volume to
detect such parameters as the number of molecules and the diffusion time . The temporal changes
in the fluorescence emission intensity is recorded which is caused by single flourophores that
pass through the detection volume. The intensity changes are quantifiable by their duration and
strength by temporally auto-correlating the recorded intensity signal. Eventually, important
biochemical parameters can be determined as the concentration, size, shape of the particle or the
viscosity of the environment changes (Lakowicz, 2006).
Fig.(?): A typical
focused by an objective
aperture) to a diffraction
collected by the same
interference filter. A pinhole
reduces out of focus light. The
photon excitation (NCBI.gov,
confocal FCS system. Laser light is
(usually with high numerical
limited spot. Fluorescence is
objective and filtered by an
placed in the conjugate image plane
pinhole is usually omitted in twoFigure 2).
FCS is a sensitive
form of analytical tools due to
the fact that it is able to observe a small number of molecules that is nanomolar to picomolar
concentrations in a small volume. This in turn makes FCS the perfect method to provide
quantitative answers on diffusing molecules from within unperturbed compartments such as
cells. FCS was developed in the early seventies as a special case of relaxation analysis. Classical
relation methods induce a certain level or kind of external perturbation like pressure or
temperature jumps to a reaction system and records information about the kinetic parameters by
observing the way the system jumps back to equilibrium. FCS just as classical techniques takes
advantage of the spontaneous minute fluctuations of physical parameters that are reflected by the
fluorescence emission of the molecules. These fluctuations are continually occurring at ambient
temperature and are represented as noise patterns of measured signal in fluorescence. This
autocorrelation analysis provides a measure for self-similarity of a time series signal that
describes the persistence of the information carried . The information processes governing the
molecular dynamics can thus be derived from temporal patterns display by fluorescence
fluctuations decay and arise (Rigler& Elson, 2001).
Fluorescence Cross-Correlation Spectroscopy (FCCS) is a daughter technique that
correlates signals originating from two different fluorophores detected in two channels with each
other. When two different spectral fluorophores are attached to two molecules they form a dual
color FCCS results. This information of the degree of coinciding appearance in the optical
volume is used to learn about the degree of interaction between fluorophores. FCCS therefore
offer binding kinetics in unperturbed systems and also in low molecular concentrations in
solutions(Lakowicz, 2006).
The History of Fluorescence Correlation Spectroscopy
Almost 40 years since its introduction FCS has evolved from a mysterious and difficult
measurement to a technique that is routinely used in the research technology. FCS value in
biological and physical sciences consists in the measurements that it makes possible and the
concepts that it illustrates and that form its basis. FCS provides the window for the field of single
molecule measurement and microscopic world (Gräslund, Rigler&Widengren, 2010).
FCS was first introduced by Madge, Elson and Webb in 1972, where it was applied to
measure diffusion and chemical dynamics of DNA-drug interaction. The term FCS was coined
by the Webb lab. The main breakthrough of the technique was the introduction of the confocal
optics by Rigler and co-worker in the early 1990’s which resulted in increased sensitivity to
sample fluorescence at the single molecule level. These pioneering studies were then followed by
a number of other applications by many different groups describing translation and rotational
mobility in two or three dimensions, attempting to determine the particle concentration even in
the cellular environment. These early measurements suffered from poor signal to noise ratios,
which was mainly because of the low detection efficiency, insufficient background suppression
and large ensemble numbers (Magde, Webb &Elson,1978).
Principles and theories of Fluorescence Correlation Spectroscopy
FCS Experiments commonly involve sample volumes as low as a few microliters and
usually work best in nanomolar concentrations of proteins. The measurements can be performed
in solutions and living cells. FCS is based on the analysis of fluctuations of fluorescence. The
molecules typically originate from Brownian motion , the random motion of particles suspended
in a fluid (a liquid or a gas) resulting from their collision with the quick atoms or molecules in
the gas or liquid, of dye labeled molecules through small laser spot. These molecules stay within
the laser spot depending on their size and if a small dye tagged molecule binds to a large one it
emits photons and slows down during its diffusion time(Rigler& Elson, 2001).
A sensitive detector records single photons emitted by the molecule of dye. Correlation
functions are applied to extract information about the number of molecules (concentration) .
Physical modes are fitted to the correlation data to quantify the information on the source of the
fluctuations. (Rigler& Elson, 2001).
When one detector and one type of fluorescent dye are used the method is known as autocorrelation. To increase the flexibility of the method, two dyes and detectors are used and the
method is called cross-correlation (Rigler& Elson, 2001).
The principles of FCS are marked by different stages in the exploitation of fluctuations,
as identified and explained below:
1. Source of Fluctuations
This is free diffusion which is the movement of small particles in a solution in Brownian
motion. The molecule tagged with a fluorescent dye diffuses through a small laser spot while
emitting a burst of photons(Rigler& Elson, 2001).
2. Create a Small Measurement Volume
FCS is based on the measurement of fluctuations of different samples in solutions. The
fluctuation signal is higher with a lower number of molecules. To measure higher concentrations
a measurement volume as small as possible is created. The measurement of volume is created
using different methods like Multi-photon excitation and confocal volume (Rigler& Elson,
2001).
3. Detect Single Photons
While passing the measurement volume, the chromophore attached to the protein of interest
emits very few photons. To detect the photons very sensitive photon counting detectors are
required. Typically Avalanche Photo Detectors (APD) are used (Rigler& Elson, 2001).
4. Calculate Correlation Function
The auto-correlation function transforms the data from the measured time domain to the
correlation time domain. To calculate the auto-correlation function, one compares the measured
data with a time-shifted version of itself. If there is no time-shift and both data traces are
identical then correlation is high. If the shift is large and the two traces are very different, then
correlation is low (Rigler& Elson, 2001).
Theoretical concepts of FCS
a. Autocorrelation analysis
Autocorrelation analysis is performed when the focus is on one species of fluorescent
particles. Fluctuations in the fluorescence signal are quantified by temporally auto correlating the
recorded intensity signal. In principle, this autocorrelation routine provides a measure for the
self-similarity of a time signal and highlights characteristic time constants of underlying
processes (Schwille&Haustein, 2004).
b. Cross-correlation analysis
In autocorrelation analysis, one effectively compares a measured signal with itself at
some later time and looks for recurring patterns. In cross-correlation analysis two different
signals are correlated and thus a measure of crosstalk is obtained. It involves looking out for
common features of independently measured signals which help to remove unwanted artifacts
introduced by the detector and provides much higher detection specificity(Schwille&Haustein,
2004).
Fluorescence Correlation Spectroscopy and Nanoparticles
The prospects of using nanoparticles as superior sensors and labels that do not photobleach in biological and environmental studies has sparked wide spread interest in the science
community. For the application of nanoparticles in FCS, the diffusion of nanoparticles in a liquid
environment must be studied and understood. This is because when applying the correlation
techniques, the diffusion constants of nanoparticles are extracted from solutions mainly using the
FCS. The diffusion constant is a direct measure of the total nanoparticle size, which includes the
inorganic core and the organic capping material. FCS measures spontaneous intensity
fluctuations which in the case of nanoparticles are caused by small deviations from the
equilibrium which in turn are caused by nanoparticles entering and leaving the detection area.
Just like in the ordinary FCS, FCS that use nanoparticles obtain the largest fluctuations when
there are only few molecules or particles in a small detection volume with the ultimate limit
being a single molecule or particle at a given time. The sufficient signal to noise for a single
molecule or particle in FCS can be achieved through minimizing the detection volume by
focusing a laser beam to a diffraction limited spot combined with high quantum yield
photodetectors (Tetin, 2013).
FCS can be used to measure the diffusion of nanoparticles to understand their mobility
under different conditions like different shapes and core size, an applied external field, or
varying surface capping materials. The hydrodynamic ra …
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